Method for selecting antigen recognizing moieties having specificity for a target structure

Abstract

The present invention provides an in-vitro method that allows to assess specificity as well as tissue cross reactivity of novel binders. i.e. antigen recognizing moieties such as antibodies or antigen binding fragments thereof, wherein said binders may be beneficially used in e.g. CAR T cell therapy.

1 . An in-vitro method for selecting antigen recognizing moieties having specificity for a target structure, the method comprising
 a) providing an aggregate comprising spatially separated units comprising
 i) first units that present said target structure, or 
 ii) first units that present said target structure and second units that do not present said target structure 
   b) contacting said aggregate with a first conjugate comprising a fluorescent moiety and a first antigen recognizing moiety that can bind to said target structure,   c) removing unbound first conjugate from said aggregate of step b) and determining
 i) the intensity of the fluorescence signal emitted by the fluorescent moieties of the first conjugate present in/on each unit of said aggregate, and 
 ii) the spatial distribution of the intensities of the fluorescence signals emitted by the fluorescent moieties of the first conjugate present in/on the units of said aggregate, thereby identifying a pattern reflecting the presence of said first conjugate in/on units of said aggregate, 
   d) repeating steps b) and c) at least once by erasing the preceding fluorescence signal emitted by the fluorescent moieties of the preceding conjugate with at least one further conjugate comprising a fluorescent moiety and a further antigen recognizing moiety that can bind to said target structure, wherein said first conjugate or one of said at least one further conjugates is a positive reference   e) identifying and selecting from the conjugates of step b) to d), that are not the positive reference, the conjugates with antigen recognizing moieties that have an at least 45% overlap of the pattern as determined in step c) ii) relative to the pattern of the positive reference.   
     
     
         2 . The method of  claim 1 , wherein said target structure is a target antigen expressed on the surface of a target cell,
 and wherein said first units of said aggregate comprise individual units comprising   a) target cells expressing said target antigen on the surface of said target cells, or   b) cells expressing recombinantly said target antigen on the surface of said cells, wherein said cells expressing recombinantly said target antigen are a positive control;   and optionally in addition to a) or b):   c) non-target cells or tissue that express said target antigen on the surface of said non-target cells or of the cells of said tissue;   and wherein said second units of said aggregate comprise individual units comprising non-target cells or tissue that do not present said target antigen on the surface of said non target cells or of the cells of said tissue, wherein each individual unit comprise only one type of non-target cells or one type of tissue.   
     
     
         3 . The method of  claim 2 , wherein said second units of said aggregate comprise additionally an individual unit comprising cells that are recombinantly knocked-out for said target antigen as a negative control. 
     
     
         4 . The method of  claim 2 , wherein said second units of said array comprise at least two individual units having different types of non-target cells or type of tissues. 
     
     
         5 . The method of  claim 1 , wherein said aggregate is a cluster of first and second units on one solid phase or said aggregate is collection of first and second units that are all or partly on different solid phases allowing also a timely separated processing of the units of the aggregate that are not on the same solid phase. 
     
     
         6 . The method of  claim 2 , wherein said target cells expressing said target antigen on the surface of said target cells are cancer cells or tumor tissue, and wherein said non-target cells or tissue that express said target antigen on the surface of said non-target cells or of the cells of said tissue is/are healthy tissue or cancer cells of another type of cancer than the cancer cells/tumor tissue that are the target cells, and wherein said target antigen is a tumor associated antigen (TAA). 
     
     
         7 . The method of  claim 2 , wherein said an in-vitro method additionally comprises the step of providing an immune cell with a chimeric antigen receptor (CAR) comprising
 I) an antigen recognizing moiety   II) a transmembrane domain   III) an intracellular signaling domain,   
       wherein the antigen recognizing moiety of the CAR is an antigen recognizing moiety identified and selected in step e). 
     
     
         8 . An immune cell expressing a CAR comprising
 I) an antigen recognizing moiety   II) a transmembrane domain, and   III) an intracellular signaling domain,   wherein the antigen recognizing moiety of the CAR is obtained by the method according to  claim 2 .